1. Field of the Invention
The present invention relates to a method for measuring phosphoric acid which is useful in clinical biochemical examinations and the like. More particularly, it relates to a method for measuring phosphoric acid by the use of an enzyme cycling system using a dehydrogenase together with a thio-NADP, a thio-NAD, a reduced thio-NADP or a reduced thio-NAD as a coenzyme, which permits measurement of phosphoric acid in a wide concentration range from a low concentration to a high concentration.
2. Description of Related Arts
Phosphoric acid in blood is called “inorganic phosphorus” in clinical examinations and is an important inorganic substance in a living body which is controlled by parathyroid hormone and vitamin D. When the concentration of phosphoric acid in blood is too low, the synthesis of intracellular organic phosphoric acid compounds or cell functions are inhibited. On the other hand, when the phosphoric acid concentration is too high, ectopic calcification or the like is caused. Therefore, various diseases responsible for, for example, incretion or bone metabolism abnormality can be known by analogy by measuring phosphoric acid in blood.
As a method for this measurement, a molybdenum blue method, an enzyme method and the like are known. The molybdenum blue method is a method in which phosphoric acid is measured by adding molybdic acid to phosphate ions in a sample to form a hexavalent phosphomolybdic acid salt (yellow), and adding thereto a reducing agent such as ascorbic acid to form a trivalent phosphomolybdic acid salt (molybdenum blue), followed by colorimetry at 660 to 750 nm (Rinsho Kensaho Teiyo (Kinbara Shuppan), 31th Revised Edition, pp. 597-598 (1998)). The enzyme method, for example, a PNP-XOD-POD method is known as a method for measuring phosphoric acid by carrying out a reaction under milder conditions. The PNP-XOD-POD method is a method in which phosphoric acid is measured by treating phosphate ions in a sample with purine nucleoside phosphorylase (PNP) in the presence of inosine (a substrate) to produce hypoxanthine, making the produced hypoxanthine into uric acid and H2O2 by the action of xanthine oxidase (XOD), reacting H2O2 by the use of Trinder's reagent or the like to cause coloration, followed by colorimetry (Nihon Rinsho, Vol. 43, Special Fall Number in 1985, Kohan-i Ketsueki Nyo Kagakukensa•Menekigakutekikensa (Wide-ranging Blood and Urine Chemical Examinations•Immunological Examinations) (First Volume), p. 460 (1985)).
These methods, however, have a low sensitivity as a method for measuring a low concentration of phosphoric acid. The molybdenum blue method is insufficient in reaction specificity. The PNP-XOD-POD method is disadvantageous, for example, in that it is liable to be affected by reducing substances.
On the other hand, in JP-A-4-349898, phosphoric acid is measured by reacting D-glyceraldehyde 3-phosphate, thio-NAD and NADH with phosphoric acid in a sample. This method is advantageous in that phosphoric acid is measurable with high sensitivity by an enzyme cycling reaction method by utilizing the fact that thio-NAD(P)H and NAD(P)H are measurable in distinction from each other because thio-NAD(P)H has an absorbance near 400 nm and NAD(P)H has an absorbance near 340 nm. This method, however, is disadvantageous for practical purpose in that reagents used in this case are expensive, tend to become unstable when dissolved to give an aqueous solution, and have a low storability.